Bioactive peptide from marine sources.docx

Journal of Chromatography B, 803 (2004) 41–53Review Bioactive peptides from marine sources: pharmacological properties and isolation procedures Abel Aneiros∗ , Anoland Garateix Centre of Marine Bioactive Substances (CEBIMAR), Ministry of Science Technology and Environment, P.O. Box 10600, Loma y 37, Alturas del Vedado, Ciudad, Habana, Cuba Abstract Marine organisms represent a valuable source of new compounds. The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new active substances in the field of the development of bioactive products. In this paper, the molecular diversity of different marine peptides is described as well as information about their biological properties and mechanisms of action is provided. Moreover, a short review about isolation procedures of selected bioactive marine peptides is offered. Novel peptides from sponges, ascidians, mollusks, sea anemones and seaweeds are presented in association with their pharmacological properties and obtainment methods. © 2003 Elsevier B.V. All rights reserved. Keywords: Reviews; Isolation; Marine sources; Peptides, bioactive Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Cyclic peptides and depsipeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Bioactive peptides from sponges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Bioactive peptides from Ascidians . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Bioactive cyclic peptides from Mollusks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Isolation methods of cyclic peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Linear peptide toxins from Conus and sea anemones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45 3.1. Conus toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Sea anemone linear polypeptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Channel toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Cytolisins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Isolation of toxins from Conus and sea anemones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Isolation of cytolysins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .50 4. Peptides and proteins from seaweeds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Isolation of phycobiliproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Concluding remarks. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41 42 42 43 44 45 45 46 46 48 49 50 50 51 51 1. Introduction It is a real fact that the importance of marine organisms as a source of new substances is growing. With marine species comprising approximately a half of the total global ∗ Corresponding author. Tel.: +53-7-881-1298; fax: +53-7-881-9300. E-mail address: [email protected] (A. Aneiros). 1570-0232/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jchromb.2003.11.005 Geodiamolides A–G (Fig. Aneiros. Sponges have been traditionally known as a source of novel bioactive metabolites like terpenoids. nucleoside derivatives and many other organic compounds. A review of cytotoxic peptides from sponges [6] describes some of the discoveries in this field at that time. Bioactive peptides from sponges Sponges are a large and diverse group of colonial organisms that constitute the phylum Porifera with thousands of different species extensively distributed from superficial waters near the sea shores up to deep waters of the ocean. an anticancer compound [4]. Active peptides from sponges most of them with unique unprecedent structures in comparison with these kind of compounds from other sources are often cyclic or linear peptides containing unusual amino acids which are either rare in terrestrial and microbial systems or even totally novel. and it has been classified as the largest remaining reservoir of natural molecules to be evaluated for drug activity [2]. Theonellamides A–F are also a group of cytotoxic compounds isolated by Matsunaga et al. antimicrobial. the sea offers an enormous resource for novel compounds [1]. Polydiscamide A. macrolides. Phakellistatin 2 showed potent activity against P388 murine leukemia cells (ED50 = 0. and the similar peptide Jaspamide are also a group of cytotoxic peptides in which three amino acids form a cyclic peptide with a common polyketide unit. These facts introduce marine peptides as a new choice for the obtainment of lead compounds for biomedical research.1. because of the similarity of some of these molecules to marine microbial metabolites. Synthetic analogues of the C-nucleosides Spongouridine and Spongothymidine isolated from a Caribbean sponge led later to the development of Cytosine Arabinoside. Discobahamin A and B are two bioactive antifungal peptides evaluated as inhibitors of the growth of Candida . 1. polyethers. Discodermin A. Basic structure of Geodiamolides A–F peptides where X and R represent different atoms or radicals. Garateix / J. One of the first novel peptides isolated from sponges were the cell growth inhibitory tetradecapeptide Discodermins [5]. being actually a well established sector in the research of marine natural products. A very different kind of substances have been obtained from marine organisms among other reasons because they are living in a very exigent. Discodermins A–H and the structural related Polydiscamide A. They are all cytotoxic and were tested against P388 murine leukemia cells. 2. obtained from sponges of the genus Discodermia are a group of cytotoxic peptides containing 13–14 known and rare amino acids as a chain. alkaloids. and aggressive surrounding very different in many aspects from the terrestrial environment. A549 human lung cell line or by the inhibition of the development of starfish embryos with IC50 values from 0. it is suggested that the origin of some of these peptides would be associated with a microbial source or with symbionts within the sponges. but also against different melanoma cell lines. and its derivatives were proposed as antibacterial and antitumor agents [11]. with a macrocyclic ring constituted by lactonization of a threonine unit with the carboxy terminal. Cyclic and linear peptides discovered from marine animals have increased our knowledge about new potent cytotoxic. Cyclic peptides and depsipeptides 2. Fig. B 803 (2004) 41–53 biodiversity. 1) initially isolated from the Caribbean sponge Geodia sp. [10] with novel amino acid residues and complex bicyclic macrocyclic rings. was further studied recently [7] showing a permeabilizing effect on the plasma membrane of vascular smooth muscle cells and tissues. The discovery of the bioregulatory role of different endogenous peptides in the organism as well as the understanding of the molecular mechanisms of action of some new bioactive peptides obtained from natural sources on specific cellular targets. the attention has been directed also to the search of bioactive peptides from sponges.42 A. Chromatogr. Recently marine peptides have opened a new perspective for pharmaceutical developments [3]. A novel peptide.34 g/ml). This non exhaustive review is an intent to systematize some of the recent and novel information in this field. and also frequently containing uncommon condensation between amino acids.02 to 20 g/ml. competitive. a situation that demands the production of quite specific and potent active molecules. and many other properties with novel chemical structures associated to original mechanisms of pharmacological activity. Arenastatin A [8] a cyclic depsipeptide from Dysidia arenaria is a extreme potent cytotoxic compound with a IC50 of 5 pg/ml against KB cells. ion channels specific blockers. More recently. Phakellistatins [9] are a group of proline rich cyclic heptapeptides. As it was demonstrated later. A. contributed to consider peptides also as promising lead drug candidates. New members have been added lately to the list of bioactive peptides from sponges and many of them were evaluated as cytotoxic exhibiting antitumor and antimicrobial activities. 7 ng/ml). Mapacalcine is suggested as a specific inhibitor of a new type of calcium current. Cyclotheonamides A and B [28] are potent antithrombin cyclic peptides which strongly inhibited various proteinases. Theonellapeptolides. B 803 (2004) 41–53 43 albicans. Theonella sp. The depsipeptides Halicylindramides A–C [13] and D and E [14] with antifungal and cytotoxic properties (against P388). Some other peptides have been isolated from marine bacteria. Didemnin B (Fig. Didemnin B together with some of the more promising marine antitumor candidates was evaluated .A. Other novel peptides. hepatotoxin. Additionally other studies indicate that different compounds isolated from marine macroorganisms like sponges and tunicates may be produced from dietary or by microorganisms like bacteria or symbiotic blue-green algae [31]. Three new antifungal cyclic peptides with unprecedented amino acids. 2) was the most prominent member of the family with the most potent antitumor activity (in vitro L1210 IC50 = 2. particularly thrombin. from the Philippines [15]. Further antifungal cyclic peptides from sponges are the Aciculitins A–C [16] and the Theonegramide [17]. The potential of marine microorganisms for the obtainment of lead molecules is clearly immense due to other reasons. antiviral and immunosupressive activities primarily isolated from the Caribbean tunicate Trididemnum solidum. as well as neurotoxins. Many of these compounds have potent activities not always clearly related to their in situ role. A bioassay guided fractionation of two species of marine sponges from the Pacific Ocean [20]. the feasibility to cultivate in laboratory conditions and to manipulate these initial sources of bioactive natural products.g. Other examples are the Haligramides A and B. Didemnins are a family of depsipeptides with antitumor.064) from Cliona vastifica [29] which selectively blocked a non-L-type new calcium conductance characterized in mouse duodenal myocytes with an IC50 value of approximately 0. 2. In another study. Other examples of bioactive peptides isolated from microorganisms living in association with sponges are the bicyclic glycopeptide Theopalauamide [33] and the cytotoxic halogenated hexapeptide Cyclocinamide A with a potent in vitro selective activity against Colon-38 tumor cells [34].6E)-dienoic acid moiety N-linked to a terminal glycine residue. Chromatogr. two new cytotoxic hexapeptides from the sponge Haliclona nigra [18]. Geodiamolides J–N and P represented the first examples in the Geodiamolide family in which a serine residue has been incorporated. antivirals. The isolation and structure elucidation of the first bioactive peptides isolated directly from a marine bacterium was reported in 1991 by McKee [32]. and Cyclotheonamides. and the biologically active peptides from marine microorganisms. isolated from the Bahamian deep water marine sponge Discodermia sp. immunosuppressive and antimicrobial agents. [12]. Theonellamide F. On the other hand. led to the isolation of different new cytotoxic peptides like the cyclic depsipeptides Geodiamolides H–N and P. This fact illustrates the need for further research into the symbiotic association within these macroorganisms and also suggest to orientate more intensively the search of these compounds also directly to the marine microorganisms. e. Examples are antitumorals. Garateix / J. and Mozamides A and B from a theonellid sponge collected in Mozambique [19]. Keramamides B–D were also isolated from sponges of the same previously mentioned genus Theonella [26] as well as Orbiculamide A [27] cytotoxic against P388 murine leukemia cells (IC50 = 4. Other cytotoxic and antiproliferative peptides from sponges are Hemiasterlin [21]. were obtained from the Japanese marine sponge Halichondria cylindrata. Microspinosamide inhibited the cytopathic effect of HIV-1 infection in an XTT-based in vitro assay. 3-dihydroxy-2. in many cases are unclear [30]. that is the first naturally occurring peptide containing a beta–hydroxy-p-bromophenylalanine residue.6. Bioactive peptides from Ascidians Cyclic and linear peptides and depsipeptides with novel structures and biological functions have been discovered also in ascidians usually called tunicates. Other active peptides also isolated from members of the same genus are the Swinholide A. and the previously underscribed 2. Aneiros. A. Bistheonellides. However up to now there is no comparison with the amount of bioactive peptides isolated from marine macroorganisms. new polypeptides from sponges acting on membranes of excitable cells have been also useful to discover new targets for physiological mechanisms. Onnamide A. Microsclerodermins C–E were isolated from two species of sponges. The cyclic depsipeptides Papuamides A–D [24] isolated from sponges of the genus Theonella. and Microscleroderma sp. Another anti-HIV candidate from the sponge Sidonops microspinosa is the Microspinosamide [25] a new cyclic depsipeptide incorporating 13 amino acid residues. Halicylindramide D is a tridecapeptide also with antifungal and cytotoxic properties. HIV-inhibitory peptides from sponges constitute a recent discovery.2.8-trimethyldeca-(4Z. first identified in duodenal myocytes.5 ng/ml) [36] and it was the first marine natural product evaluated in human clinical trials. Halicylindramides A–C are tetradecapeptides with an N-terminus blocked by a formyl group and the C-terminus lactonized with a threonine residue. Milnamide A [22] and Jasplakinolide [23]. containing a number of unusual amino acids are also the first marinederived peptides reported to contain 3-hydroxyleucine and homoproline residues.2 M. but later obtained from other species of the same genus [35]. Papuamides A and B inhibited the infection of human T-lymphoblastoid cells by HIV-1 sub(RF) in vitro with an EC50 of approximately 4 ng/ml. while Halicylindramide E is a truncated linear peptide with a C-terminal amide. and cardiac stimulants. Multiple-step purification procedure led to the new dimeric peptide Mapacalcine (Mr 19. The origin and role of bioactive peptides inside the sponges. that is retained in high salinity (200 mM NaCl). but this is only a preliminary supposition. Dolastatin 10 has been evaluated with promising perspectives in a phase I clinical study in patients with solid tumors. New classes of anticancer drugs candidates. A. 3. Styelin D exhibited activity against Gram-negative and Gram-positive bacteria. Another recently discovered cytotoxic peptide from an ascidian is Styelin D [42]. one proline. Didemnim B from the tunicate Trididemnum solidum. 3) when isolated in 1987 [46] was reported as the most active anticancer natural substance at that time with a ED50 of 4. a 32-residue C-terminally amidated antimicrobial peptide. Nordidemnin N. and novel and unusual amino acids. M. and Epididemnin A. one valine. two phenylalanine residues.6 × 10−5 g/ml against the P388 cell line. in this case highly compact and stable linear peptides. Tamandarins A and B are also two cytotoxic depsipeptides recently discovered from a marine ascidian of the family Didemnidae [43]. isolated from blood cells (hemocytes) of the ascidian Styela clava. Lissoclinamides constitute another large family of cyclic peptides [40] isolated from ascidians (Lissoclinum patella) showing relevant antineoplastic as well as other pharmacological properties against human fibroblast and bladder carcinoma cell lines. MEL-20. Dolastatins are a group of cyclic and linear peptides isolated from the marine mollusk Dolabella auricularia. This author indicates that these kind of peptide antibiotics from marine organisms could be useful for the development of topical microbicides in conditions of physiological or elevated NaCl concentrations. Accumulated evidences support the fact that Opisthobranchia mollusks have developed very powerful chemical defenses [44]. isolated from Pleurobranchus forskalii [50]. The molecule contains extensive post-translational modifications. More than a dozen of Dolastatins have been isolated up to now. The cytotoxicity of Tamandarin A was evaluated against various human cancer cell lines and shown to be slightly more potent than Didemnin. Recent studies have shown for example that the depsipeptide Dolastatin 11 arrests cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network and induces hyperpolymerization of purified actin [49]. The pentapeptide Dolastatin 10 (Fig. Subsequently its noticeable antitumor activity was well documented in various in vitro and in vivo tumor models [48]. B 803 (2004) 41–53 Fig. as well as twenty-two new Didemnin analogues were prepared semisynthetically and their biological activities evaluated. . Dolabella auricularia is a mollusk devoid of shell and for this reason initially appears to lack defenses against predators.44 A. Some of them are Didemnins X. Chromatogr. including cytotoxicity and antiviral and immunosuppressive properties [39]. also cyclic depsipeptides related to Didemnins have been later isolated from extracts of this tunicate. Dolastatin 10 from the sea hare (mollusk) Dolabella auricularia. but Dolastatin 11 was about three-fold more cytotoxic than Jasplakinolide in the cells studied. The effects of Dolastatin 11 were most similar to those of the sponge-derived depsipeptide Jasplakinolide. Bioactive cyclic peptides from Mollusks Cytotoxic cyclic peptides have been also found in mollusks. According to structural characteristics. with prominent cell growth suppressing activity. as an antiproliferative agent against human prostatic cancer cell lines [37]. Keenamide A exhibited significant activity against the P-388. Y. as it will be shown later in Section 3. A-549. cytotoxic compounds from marine Fig. The study of the cytotoxic mechanism of action of members of the family of Dolastatins exhibit particularities. 2. From mollusks it has been also isolated another prominent family of peptides. Some members of the family contain an oxazoline ring. and dengue virus. known as conotoxins but with specific actions on ion channels and membrane receptors of excitable cells. 2. and thiazole and/or thiazoline rings [41]. Another study showed that Dolastatin 10 inhibits tubulin polymerization and tubulin dependent GTP hydrolysis [47]. Garateix / J. parainfluenza. Different new members of the family. and HT-29 tumor cell lines. Another new cytotoxic cyclic hexapeptide from a marine mollusk is the Keenamide A. among others [38].3. Didemnim A and B also show antiviral activity against different kinds of virus like herpes simplex. Neurotoxic side effect for this compound was also detected and in this sense some caution in the use of it in the clinic was suggested.1. isolated from marine organisms. The earliest information about the isolation of some of these compounds was reported by Pettit in 1981 [45]. Aneiros. At concentration levels of pmol/ml Didemnin B was more effective in the inhibition of prostate cancer cell proliferation than Taxol. have been shown to possess powerful cytotoxic activity against multiple tumor types. sponges and mollusk. [60] the list of macromolecular targets of these peptides includes six types of ion channels and receptors: acetylcholine receptors ( -. As the use of venoms is the strategy of different species to survive in a specific environment it is not surprising that the components of these venoms might exert very specific and potent effects. According to Cruz et al. and a separation step in a Sephadex LH-20 column eluted with methanol–water. usually include initial extraction with methanol (MeOH). They consist of a mixture of peptides. of relatively short strings of amino acids and are rich in disulfide bonds. A. peptidic toxins can adopt relatively stable compact conformations. a cyclodepsipeptide from the mollusk Onchidum sp. A. The methanol extract is concentrated in vacuum and the resulting viscous concentrate is partitioned between 10% aqueous methanol and hexane. commonly known as toxins.62]. D. and later with dichloromethane (CH2Cl2).54]. they seem to be very potent and additionally for the biosynthesis of proteins the required machinery is already present in animals [55]. The methanolic portion is then extracted with carbon tetrachloride (CCl4). NMDA receptors (conantokins). In conformity with Schmitz et al. a typical procedure of a bioassay guided isolation applied to the obtainment of Onchidin B. B 803 (2004) 41–53 45 organisms include very different kind of metabolites like macrolides. The presence of disulfide bonds seems to lock the peptide into a relatively rigid shape. Aneiros. As many neurological disorders are the result of mutations at this level (termed “channelopathies”) such mutant ion channels represent an important therapeutic target for the discovery of new drugs. Dolastatins 3. and D. In addition Conus venoms also contain a heterogeneous group of peptides that are not disulfide rich (e. just before the HPLC chromatography on the reversed phase C18 column [53.1. the discovery of new conopeptides having specific structural and biological features is enhancing. According to Olivera [57] these peptides take part in the defence. Chromatogr.and -conotoxins) calcium ( -conotoxins) and potassium (k-conotoxins) channels. CCl4. which probably increase the binding ability of conotoxins to very specific targets [59]. Garateix / J. O. Dehydrodidemnimn B. The majority of these peptides consist of about 8–35 amino acids in length [58].and -conotoxins). partitions of this extract with organic solvents of increasing polarities to render diverse organic fractions. 10. The -conopeptides represent the best structurally studied class and they act as antagonists of the nicotinic acetylcholine receptors (AchRs) [61. and chromatographic steps on silica and Sephadex LH-20 columns. terpenes. E. therapeutic. Each organic layer is concentrated in vacuum to yield the hexane. However. large polypeptides (>10 kDa) or small molecules such as biologically active amines. 2. they are being used as useful chemical tools in the field of scientific research to study the molecular mechanisms underlying different functions. The use of toxins is an expanding field and up to the moment they have been used as lead structures for diagnostic. For this same reason animal venoms are for scientists a source of interesting bioactive molecules . According to Arias and 3.A. X. As an example. B. [51] the most active cytotoxins (ED50 < 10−3 g/ml) are the depsipeptides. and the use of reversed phase C18 HPLC for the final purification. and CH2Cl2 extracts. Conus represent a valuable source of neuropharmacologically active peptides named conotoxins. vasopressin (conopressins) and neurotensin (contulateins) receptors (Table 1). They include Didemnims B. 11. Linear peptide toxins from Conus and sea anemones The animals’ use of venoms for feeding. Several studies have shown that some of the most active components of venoms are of proteinic nature probably because of different reasons. making them resistent to enzymatic actions and exerting very specific effect at the level of their target receptors because of their complex structure [3]. The relatively small size of these peptides represents a great advantage for their chemical synthesis [60]. defence and other interspecies interactions results from an evolutionary fine process that has taken place in different taxa along millions of years. nitrogen including compounds like complex alkaloids. and other purposes. [52] include an initial extraction from the freeze-dried animal with methanol. C. because of structural foldings. and mollusks. 15 and Geodiamolides A. The CCl4 extract was submitted to flash silica gel column chromatography eluted with a gradient from dichloromethane to methanol resulting in five fractions. Moreover. the conantokins). 3. and many others.g. The ion channels are a common target for many peptide toxins. Moreover. Isolation methods of cyclic peptides Purification procedures of these kind of cyclic peptides and depsipeptides isolated from sea animals like ascidians. Fraction two was further purified on a reversed phase C18 HPLC and resulted in purification of Onchidin B. sodium ( -. Their large surfaces allow more bonding contact with their target receptors. prey capture and some other biotic interactions.4. Conus toxins There are approximately 500 species of predatory cone snails within the genus Conus (Phyllum Mollusca) and it has been estimated that the venom of each Conus species has among 50 and 200 components [56]. However among the most active of them are the cyclic peptides and depsipeptides or linear peptides bearing uncommon amino acids isolated from sponges. tunicates. Variants of this procedure additionally include for example steps of desalting of organic (alcoholic) fractions with the resin Amberlite XAD-2. O-Conotoxins MrVIA and MrVIB isolated from C. They offer an interesting alternative in the treatment of epilepsy. They include -conotoxins GVIA and MVIIA from C. Four new -toxins were isolated from the venom of C. Specifically -conotoxins. -Conotoxin-GmVIA. characteristic of the Coelenterate Phyllum.2. synthesized and developed a structure-activity study of analogs to SNX-111. pain. scorpions and snakes [70]. 3. marmoreus potently block the sodium conductance in Aplysia neurons. geographus and C. Serotonin receptors are another molecular target for conotoxins and specifically -conotoxin GVIIIA selectively inhibits the 5-HT3 receptor [71]. It slows down the inactivation process of this current and induces action potential broadening. As it was indicated by Olivera [57] these peptides were originally purified following their characteristic to induce a sleep-like state in mice under 2 weeks of age.. Although these peptides exhibit a pharmacological action which is similar of the -conotoxins. catus and their potencies in different bioassays were compared [67]. The synthetic peptide SNX-111 corresponds to the sequence of an -conopeptide MVIIA obtained from the venom of Conus magus and it acts as a highly potent and selective antagonist of N-type calcium channels [68]. The overall fold of -conotoxin is similar to that of calcium channel-blocking -conotoxins but differs in this aspect from potassium channel-blocking toxins from sea anemones. phospholipases . For instance. Sea anemone linear polypeptides 3. The toxicity of these animals is in part associated to the presence of nematocysts. respectively. Different types of conotoxins act on the voltage-sensitive sodium channels.2. It acts specifically on molluskan sodium channels [64]. All conantokins are linear peptides except the conantokin isolated from C. The best studied of these compounds are -conotoxins which act as selectively blockers on vertebrate skeletal muscle subtypes of voltage-dependent sodium channel [58]. From nematocyst rich tissues. hypertension. induces convulsive-like contractions when injected into land snails but has no detectable effects in mammals. there have been isolated few new conotoxins which target at different sites.1. they are structurally unrelated [65]. Because of the therapeutical potential of this type of neurotoxins in the management of severe pain they are of great interest. Parkinson’s disease. purified from Conus gloriamaris. From the venom of Conus purpurascens the first cone snail toxin that blocks potassium channels ( -conotoxin PVIIA) was isolated. They represent a novel class of NMDA receptor antagonists that can modulate CNS excitability. Conantokins are a specific class of linear peptides which inhibit N-methyl-d-aspartate (NMDA) receptors This kind of receptors plays an important role in the pathophysiology of central nervous system (CNS) disorders. Neurex Corporation presented a patent (No. In addition. cancer and also as muscle relaxants [61]. whereas in mice older than 3 weeks a hyperactivity is observed.46 Table 1 Types of Conus peptides and molecular targets Molecular target Acetylcholine receptors A. magus. secretions as well as different parts or the whole body from sea anemones have been obtained different compounds of proteinic nature including pore-forming toxins or cytolisins [72–74]. A. selective for calcium channels. The team of Nadasdi et al. Garateix / J. a specialized stinging organell. were purified using their ability to produce a shaking behavior in mice as it was described by Olivera [66]. Aneiros. O-Conotoxins include a family of peptides which block sodium currents of voltage-sensitive sodium channels. radiathus which has a three amino acid disulfide bridged loop near the C-terminus [61]. pain. 5364842) [69] for -conopeptides or derivatives (SNX-111 or SNX-185) to be used as potent analgesics. Channel toxins Sessile animals like sea anemones have developed along the evolution specific chemical mechanisms for the complex interactions in which they participate in the marine environment. B 803 (2004) 41–53 Cysteine arrangement CC–C–C CC–C–C–C–C CC–C–C–CC CC–C–C–CC C–C–CC–C–C C–C–CC–C–C C–C–CC–C–C C–C–CC–C–C Linear C–C Linear Class -Conotoxins A-Conotoxins -Conotoxins -Conotoxins O-Conotoxins -Conotoxins -Conotoxins -Conotoxins Conantokins Conopressin Contulateins Mode of action Competitive inhibitor Competitive inhibitor Noncompetitive inhibitor Block current Block current Delay inactivation of Na+ channel (site VI) block current of specific subtypes Blocker Inhibitor Agonist Agonist Prototypical example -GI A-EIVA -PIIIE -PIIIA O-MrVIB -TxVIA -MVIIA A-SVIA Conantokin-G Conopresin-S Contulakin-G Sodium channel Calcium channel Potassium channels NMDA receptor Vasopressin receptors Neurotensin receptors Blanton [63] these neurotoxins are capable to discriminate between muscle and neuronal type AchRs and even among distinct AchR subtypes and the binding toxin-receptor shows specific characteristics It has been postulated that these peptides could be of interest in the treatment of anxiety. Chromatogr. -Conotoxins which act on neuromuscular sodium channels might be useful to treat neuromuscular disorders [61]. among others. stroke and Parkinson’s disease [61]. Many of these toxins induce a positive inotropic effect in different mammalian heart preparations and an increase in the action potential (AP) duration. however different residues that seem to play a role in the interaction with toxin receptor such as Arg-12. other neurotoxins [84] and even proteinase inhibitors [85. ApC from A. The studies on sea anemones toxins were started by Shapiro [89] and Béress and Béress [76] who isolated toxins from Condylactis gigantea and Anemonia sulcata. 4. Other toxins have been isolated which do not fall nearly into one of these classes. or Van der Waals interactions [98]. As it can be seen a considerable similarity exist in the amino acid sequence among them. 3. hydrogen-bonding. dashes represent gaps. sea anemone toxins are basic proteins that bind across to the IVS3–S4 loop through electrostatic.97]. According to sequence homologies sodium channel toxins can be classified as types I and II [78]: · Type I toxins are represented by ATXI. basic amino acids of toxins and acidic residues of the domains I and IV of the sodium channel alpha subunit have been implicated in toxin binding. Polypeptides in the MW range ≥ 10 000. [93]). 2. a different type of polypeptide neurotoxins acting on the voltage-dependent K+ channels was reported [79–83]. Basic polypeptides with MW ≤ 3000. K+ channel inhibitors [79–83].A.8 Fig. According to the molecular weight (MW) and some pharmacological properties. and IVS5–S6 loops of the ionic channel [98]. At the right column the percentage of identity in comparison to BgII (modified from Goudet et al. 4. the exact origin of both neurotoxins and cytolysins is not quite clear and others sources besides nematocysts seem to contribute to the toxicity of sea anemones [74]. Chromatogr. one of them take into account their sequence homologies and the second one is according to the specific binding site and to their specific physiological effect in the sodium channel. Their main effect is to delay sodium channel inactivation which leads to neurotoxic (repetitive firing) and cardiotoxic (arrhytmia) effect. Lys-49 and Lys-37 were identified [92. AftI and AftII from Anthopleura fuscoviridis. xantogrammica. Aneiros. gigantea inhibit the Na+ channel inactivation [90. Comparison of the sequences of some anemone toxins: BgII and BgIII from Bunodosoma granulifera ApA from Anthopleura xanthogrammica and ATXII from Anemonia sulcata.100. B 803 (2004) 41–53 47 [75].94].86]. In general. Polypeptides in the MW range between 4000 and 6000. In general. These compounds show mainly cytolytic activity. These two types of toxins are cross linked by three disulfide bridges. have molecular weights about 5 kDa and show the same electrophysiological activity. Garateix / J. Identical amino acids are indicated with a black background. respectively. Voltage-dependent sodium channels constitute an important target for different groups of neurotoxins which have contributed to the structural and functional characterization of this channel [95]. Chemical modification studies of sea anemone toxins were carried out by several laboratories to determine the role of different amino acids in the channel binding but do not exist a general conclusion about the role of each residue. Nevertheless. Arg-14. At least six different neurotoxin receptor sites have been described for the mammalian Na+ channels and sea anemone peptide neurotoxins and -scorpion toxins share receptor site 3 on sodium channels [96.91]. helianthus and toxins from Heteractis sp. BgII BgIII ApA ATXII 1 10 20 30 40 50 % id. · Type II toxins include ShI from S. These studies led to the discovery of new compounds capable to act at the level of voltage activated sodium channels. 4. Different authors have suggested that it is possible that the cytolisins and the toxins that target ionic channels together act synergistically [87]. It has not been corroborated subsequently if all the four groups of polypeptides are present in each sea anemone specie. GASCRCDSDGPTSRGNTLTGTLWLIG-CPSGWHNCRGSGPFIGYCCKQ--GASCRCDSDGPTSRGDTLTGTLWLIG-RCPSGWHNCRGSGPFIGYCCKQ--GVSCLCDSDGPSVRGNTLSGTLWLYPSGCPSGWHNCKAHGPTIGWCCKQ--GVPCLCDSDGPSVRGNTLSGIIWLAG--CPSGWHNCKKHGPTIGWCCKQ--- 100 97. which involves the extracellular loop IVS3–S4 and additional unidentified contacts in the IS5–S6. This group posses proteinase inhibiting activity and they show great homology with other proteinase inhibitors isolated from mammalian species. different type I sea anemone toxins are depicted. Na+ channel toxins [76–78].101]. A. that can be explained by modulation via Na+/Ca2+ exchange that results from the increased cellular Na+ load [99].92] and BgII and BgIII from Bunodosoma granulifera among others [93.9 70. In Fig. These two groups include toxins that act on the voltage-activated Na+ channel. elegantissima [90. More recently. . sulcata. ATXII and ATXV from A. Polypeptides in the MW range between 6000 and 7000. sulcata and C. sea anemone sodium polypeptide toxins were earlier classified in four groups [88]: 1. ApA and ApB from A. After that time many others polypeptide toxins have been isolated and chemically characterized from different sea anemone species which similarly to toxins from A. homologous amino acids are indicated with a gray background. There are two more recent classification of the sodium channel toxins.9 72. a well-recognized therapeutic target present in erythrocytes. Because of their action mechanism and their specificities sea anemone toxins and scorpion toxins have been used to identify the pore region and to measure the external mouth of the channel pore [102]. ShK from Stichodactyla helianthus [80]. ShK blocks different Kv1 channels at nano and subnanomolar concentrations [87. In addition ShK is a potent blocker of Kv1. The most studied actinoporins are Equinatoxins II. HmK from Heteractis magnifica [82] and AeK from Actinia equina [83] which are structurally different in comparison with other K+ channel toxins isolated from snake. Radianthus macrodactylus [112] and Condylactis aurantiaca [111. Hemolysis has been mainly used to characterize the activity of actinoporins [119]. as a low affinity receptor [120]. The second step involves slower oligomerization in the plane of the membrane. Garateix / J. a lipid that is ubiquitous in animal cells. blockers of this channel are of interest too as potential immunosuppressants [109].105].80]. Even at 100 nM.114]. granulifera [79]. Chromatogr. cysteinless proteins with isoelectric point values mostly above nine. In comparison with group II cytolysins they possess cysteine residues and lack tryptophan. it is concluded in few seconds and is irreversible [113.1 channel [87].4 potassium channel [110]. BgK blocks K+ currents in snail neurons [106] and Kv1 channels in Xenopus oocytes almost equipotently at nanomolar concentrations [87]. and ShK did not affect the Kv3.104]. AsKS and AsKC from A. Three or four molecules oligomerise in the presence of a lipid membrane to form cation-selective pores of about 2 nm hydrodynamic diameter [117. Sticholysin I and II [73] from S. IV and V from A. . · About 20 kDa pore-forming proteins inhibited by sphingomyelin. Group 2: This is the best studied group of sea anemone cytolysins. B 803 (2004) 41–53 Concerning to the K+ channels more recently a new type of blocking toxins from different sea anemones was obtained such as BgK from B. sulcata [81]. The binding of these polypeptides to the membrane is associated with a partial conformational change of the toxin without a significant change in its secondary structure.103]. scorpion and bee venoms. equina [121]. Cytolysins isolated from T. · About 30–40 kDa cytolysins with or without phospholipase A2 activity. 3. and an RGD-motif [74]. Both BgK and ShK compete with dendrotoxin-I (from snake venom) for binding to rat brain synaptosomal membranes. magnifica [82] was reported to inhibit the binding of [125I]. They are known as actinoporins [77] taking into account that their source are sea anemones (Actinaria) and their ability to form pores in natural and model lipid membrane. The primary structure of these actinoporins is characterized by a conserved putative N-terminal amphiphilic -helix. helianthus and HMgIII from H. Group 1: Cytolysins belonging to this group have been found in Tealia felina [111]. A common characteristic of both sea anemone and scorpion potassium channel blockers is the presence of a functional diad formed by lysine and a hydrophobic residue [103.-dendrotoxin (a ligand for voltage-gated K+ channel) to rat brain synaptosomal membranes with a kI of about 1 nM blocks K+ currents through Kv1.2. This pore formation includes at least two steps: the first one involves the binding of a soluble toxin monomer to the lipid bilayer. a triptophan-rich stretch. The structural characteristics of this group of cytolysins is not well known. whereas for ShK the diad is composed by a Lys-22 and Tyr-23 [104.48 A. and suppress K+ currents in rat dorsal root ganglion neurons in culture [79. A. they potently blocked at nanomolar concentrations the intermediate conductance Ca2+-activated potassium channel IKCa1.3 potassium channel in Jurkat T-lymphocytes [108]. the potassium channel toxin from the sea anemone H.112]. In contrast.116]. sulcata the blood depressing substances BDS-1 and BDS-II are the first specific blockers for the Kv3. BgK. These sea anemone toxins are formed by about 35–37 amino acids and they present three disulfide bridge [87]. macrodactylus show antihistamine activity.2.2 channels expressed in a mammalian cell line and facilitates acetylcholine release at the avian neuromuscular junctions. They are also highly cytotoxic and lytic to a variety of cells and their vesicular organelles. They use sphyngomyelin.3 channel has been implicated in the T-lymphocyte proliferation and lymphokine production. As the Kv1. · A putative group of proteins represented at present solely by an 80 kDa Metridium senile cytolysin. They also produce significant degranulation of granulocytes and platelets. The cytolysins of this group in general are not inhibited by sphingomyelin and are less hemolytically active than the groups II and III cytolysins. human T lymphocytes and colon [107]. which eventually leads to the formation of the functional pore [115.118]. This diad of toxins residues determinants for the potassium channel interactions consist in a Lys-25 and Tyr-26 for BgK. this process is fast. Additionally. Aneiros. felina and R. They are all monomeric. Cytolisins According to their molecular weight sea anemones cytolytic polypeptides can be classified in four groups [74]: · About 5–8 kDa peptides with antihistamine activity. From A. magnifica [72]. HmK. Group 3: This group include proteins with phospholipase A2 like activity. Actinoporins are very potent toxins that affect almost all kind of eukaryotic cells. They are highly lethal in mammals and this activity is attributed to cardiorespiratory arrest and coronary vasospasm. They inhibit K+ channels by binding to a receptor site in the external vestibule of the channel occluding physically the pore and thus blocking the ionic current [102]. According to a study [126] the toxin does not show high specificity for interactions with lipids. and it was concluded that this toxin is capable of forming channels at low toxin concentrations and larger pores at high concentrations which results in lipid bilayer membrane destruction. an 80 kDa haemolytic and lethal protein from M.3. Garateix / J. sometimes with only one different amino acid in a specific position of the chain but with a dramatic influence in the pharmacological activity in contrast. [132] which indicated that the separation of the isotoxins could not be achieved even with the use of very precise gel filtration and ion exchange chromatographic techniques. Sometimes when a toxic acidic peptide is detected. dog. The same general procedure was recently applied by the same research group to isolate the new peptide mr10a from Conus marmoreus. batch-wise adsorption of the toxins on a cation exchanger followed by gel filtration on Sephadex G-50. included combinations of traditional chromatographic methods for protein group separation as size exclusion and ion exchange chromatography.128]. Thus. the employment of the research-grade Serdolit® AD-2 as an initial additional alternative of normal pressure hydrophobic adsorption step and the repeated use of the same chromatographic step or rechromatography to improve the separation. and the extensive use of all the possibilities of RP-HPLC chromatography [79. Intravenous administration of Up1 produced a fall in blood pressure in normotensive rats partially reduced by atropine. . In brief. An early now classical procedure described by Béress et al. other different crude starting extracts have been used like sea anemone secretions [79] and mainly the whole animal body extract [99]. ATX II and ATX V toxins previously isolated from A. A. The presence of isotoxins in sea anemone venoms became later a well known fact. and bee venoms as a source of toxins. B 803 (2004) 41–53 49 The venom of the sea anemone Aiptasia pallida possesses hemolytic activity which is produced by at least three synergistic venom proteins. One of these proteins is a phospholipase A2 which exists in two forms. Some of the modifications or additions later included are the use of “tentacle” ion exchangers like Fractogel®. Group 4: This group is represented by metridiolysin.81. sulcata into different isotoxins. Whole body extracts are very complex mixtures of a large number of molecules containing not only toxins but also many other components from tissues and sea water salts. Up1 is a potent hemolysin on erythrocytes of rat. The use of a RP C-18 HPLC column enabled the resolution of the sea anemone toxins from A. or TSK HPLC cation exchangers. the precise localization of sea anemone toxins inside the animal is not always clear or another origin not associated with nematocysts has been suggested. which was correlated with cholesterol contents in erythrocyte membranes [125]. The toxin exhibits preference for the lysis of horse and dog erythrocytes. comprise alcoholic extraction of the crude toxic material from freshly collected sea anemones. comprise gel filtration and reverse-phase HPLC as it was used to purify the potent sleeper peptide Conotoxin GV which contains five residues of -carboxyglutamate in a chain of 17 amino acids. isolated from the fish hunting marine snail Conus geographus [127. The general procedure used to fractionate the complex mixture of different peptide toxins contained in crude Conus venoms. the crude venom was size fractionated using Sephadex G25 with acetic acid as elution buffer. This facts led to the discovery of sea anemone isotoxins. [130] for ion channel toxins isolation from A.123]. On the other hand. which has its own peculiarities in comparison with the same process from crude snake. then ion-exchange chromatography on QAE Sephadex A25 and SP Sephadex C25 and desalting on Sephadex G25. The method described above with slight variants has been later used for the obtainment of other sodium and subsequently for potassium channel sea anemone toxins. 3. scorpion.135]. Chromatogr. which is the most frequently used initial source. sulcata [130] indicated that these toxins were not pure proteins. sulcata. with a further rechromatography in the same column to eliminate contaminants and a final purification step on a Vydac protein-SCX (strong cation exchange) column. each having microheterogeneities in the sequence. and [75]. This has to be taken into account for the isolation procedure. It also produces ileal contractions similar to acetylcholine and such action was inhibited by atropine. It is a cholesterol inhibitable cytolysin [124]. senile. with potent antinociceptive properties [129]. A very similar procedure was described for the isolation of toxins from the sea anemone Radianthus paumotensis [131]. and after lyophilization the active fraction was fractionated on a C18 HPLC column with a linear gradient of acetonitrile in trifluoroacetic acid. isolation methods of sea anemone sodium channel toxins used since the beginning. Even though that the presence of some toxins in typical structures denominated nematocysts from coelenterates is well documented in the literature [133. Isolation of toxins from Conus and sea anemones Peptide toxins with specific action on ion channels from marine animals like coelenterates (sea anemones) and mollusks (Conus) are mainly linear basic peptides.A. Aneiros. BioGel. a step of anion exchange chromatography is included in the protocol to separate basic from acidic toxins. For that reason the isolation procedure has to include steps for basic peptide purification. Its hemolytic activity is not inhibited by cholesterol [122]. Phospholipase activity was also reported in Up1. A description in details of these isolation procedures is given by Béress et al. According to Cline [123] it produces a potent positive inotropic effect of the left atria of the heart of rat and guinea pig with little or no increase in heart rate in vivo. from the crude extract of the sea anemone Urcinia piscivora [122.134]. and humans causing numerous holes and breaks on membranes as indicated by scanning electron microscopy. Sequence studies of ATX I. guinea pig. It contains 91 amino acids and two disulfide bonds. 4.1. Lectins are glycoproteins with aggregation and recognition functions in the organism that posses them. a blue-colored one (fraction A).transCeratospongamide. Because of its applications.cis-Ceratospongamide and trans. specifically in fluorescent immunoassays [149]. phycobiliproteins play an important role in the photosynthetic process of blue–green. were proposed to be useful for both the diagnosis and therapy of certain brain and central nervous system disorders resulting from malfunctions of systems controlled by the neurotransmitter -aminobutyric acid (GABA). B 803 (2004) 41–53 3. A mitogenic hexapeptide with the sequence Glu-Asp-Arg-Leu-Lys-Pro denominated SECMA 1 was purified from the alga Ulva sp. Sticholysins I and II (StI and StII) purified by Lanio et al. respectively [120]. whereas the cis. One typical procedure [154] for the isolation of these proteins and in particular of phycoerythrins includes a combination of different steps of density gradient centrifugation. Phycobiliproteins are oligomeric proteins. Phycoerythrin is the most used phycobiliprotein [148]. A. cis.146]. with a highly basic isoelectric point and molecular weight of 19 392 and 19 283 (±2) Da. In another field of application. Several of these oligopeptides were more active in competing for specific super(3)H-muscimol binding to the mammalian GABA sub(A) receptors than GABA itself.4. Ceratospongamides are two isomers of a new bioactive thiazole-containing cyclic heptapeptide. On the other hand. triquetrum lectin does not show amino acid sequence similarity with known plant and animal lectin structures [140]. red and cryptophyte algae. Peptides and proteins from seaweeds Biologically active peptides and proteins have been isolated not only from marine animals. and another step of sucrose density gradient centrifugation is used for the obtainment of two main colored fractions. PBS dissociation is later achieved. This property is specially used in the field of biotechnological applications [147] where they are used in a wide variety of biomedical diagnostic systems mainly in immunochemical methods. and Lanio et al. Garateix / J. are two cysteinless polypeptides containing a high proportion of nonpolar amino acids. a novel class of nontoxic bioactive oligopeptides. column chromatography and electrophoresis. built up from two chromophore-bearing polypeptides. Phycobiliproteins have also antioxidant properties [151] with applications in cosmetology. As it was shown before. helianthus. Isolation of phycobiliproteins Isolation methods and molecular characterization of phycobiliproteins from seaweeds are described in the literature [152. [137] by this procedure. trans. Mitogenic and antineoplastic isoagglutinin glycoproteins have been equally described from the red alga Solieria robusta [141]. and in the food industry. but also from seaweeds.cis isomer is inactive [144]. some useful proteins like lectins and phycobiliproteins (PBP) have been reported from seaweeds. absorbing them all at different wavelength of the spectrum [145. the first step is the isolation of an intact phycobilisome fraction from the crude seaweed extract containing phycobiliprotein pigments by a method [155] including disruption of cells and a discontinuous sucrose gradient centrifugation. and they have proved to be useful in the field of clinical diagnosis and other health applications. In a screening of medicinal potentialities of seaweed were discovered different proteins and peptides exhibiting bioactives properties [138]. In brief. They include three main groups of compounds: phycoerythrins (PEs) with a red pigment joined to the protein molecule.153]. demonstrating their potential as a new class of potent GABA analogs. Although in a minor scale in comparison with marine animals. Similar procedures have been also used for different authors to purify other members of the family of sea anemone cytolisins referred before. [137] to isolate protein cytolysins from the sea anemone S. the phycobilisomes (PBS). found in marine red algae and cyanobacteria [143]. which is active on human foreskin fibroblasts [142]. Aneiros. Lectins have been isolated before from primitive marine organisms like sponges [139]. They were isolated from the marine red alga (Rhodophyta) Ceratodictyon spongiosum containing the symbiotic sponge Sigmadocia symbiotica. Isolation of cytolysins Purification of cytolytic toxins or cytolysins from sea anemones aqueous extracts commonly include a first gel filtration step (usually Sephadex G50 or similar) followed by cation exchange chromatography (CM-32 cellulose or other equivalent exchanger) as essential combination to achieve the resolution of the complex crude materials. phycoerythrin has been the most studied phycobiliprotein. As an example. and the other pink-violet (fraction B). This is also the case in cyclic peptides isolated from some algae. this protocol was used by Kem and Dunn [136].trans-Ceratospongamide exhibits potent inhibition of secretory phospholipase (sPLA) expression in a cell-based model for antiinflammation (ED50 = 32 nM). fluorescent immunohistochemistry [150] and other methodologies. Chromatogr. Fractions A and B are submitted separately to chromatography on columns of the anionic DEAE-cellulose DE52 developed with a linear gradient of . Light is efficiently collected by phycobiliproteins assembled into macromolecular structures.50 A. 4. From the red marine alga Bryothamnion triquetrum a member of a new structural family of lectins was isolated. The primary structure of the B. phycocianins with a blue pigment and alophycocianins. Phycobiliproteins are spontaneously fluorescent “in vivo” and “in vitro” molecules. an origen associated with symbionts from some new metabolites isolated from marine macroorganisms is suspected. Eng. J.P.K. Amano.A.L.G.E. Toth. Allen. J. H. G. [27] N.T. New York. J. J. Kitagawa. D. P. Herald. Prod. Biol. R. Am. Int. N. Am.I. as judged by their electrophoretic composition are further purified by centrifugation on a linear sucrose density gradient. [24] P.R. Diagn. D. N. Soc. J. Sato. B.J. Faulkner. Pettit. J. . Kobayashi. Morel. Stroh. Pt. Walchli. Doubek.R. N. Rinehart Jr.A. [14] H. Mitusi-Saito. Int.R. Plenum Press. Van-Soest.A. Li. T. 63 (1998) 1254.E. Durso. Sci. Med. van den Brenk. Craig. J.E. 1. Gunasekera. M. Osaki. Abst.F. R. G. T.K.J. 112 (1990) 7053. Am. Andersen. Prod. T. Plenum Press. 26 (1985) 855. D. Shigematsu. K. D. [19] E. Chem. G. Canonico. 270. Fusetani. 35 (1994) 7969. 7 (2000) 947.. E. N. J.K. Lehrer.D. H. Moore. Because of the peculiarities of the life in the sea.L. Matsunaga.L. References [1] [2] [3] [4] [5] [6] [7] D. M. J. Med. Li. Williams. D. Y.L.J.A. T. Qar. Mironneau. Nakamura. 113 (1991) 7811. 1993. S. A. 1993. A. 113 (1991) 7818. Clin. G. McCarthy.. Schmidt. M.P. Pannell. 277. Odaka. 51 (1991) 1. 53 (1990) 771. p. [40] F. Prod. P. Nat. Bowden. J. Soc. 59 (1994) 2932. Chem. Faircloth.D.R. Am. Marine Biotechnology. Wang. Boyd. Sci. H. 22 (1982) 696. 275 (2000) 38417. K. [18] M. N. Fusetani. 121 (1999) 5899.M.S. Chem. Geldof. Fenical. Nat.). J. [39] R. [17] C. Sautierre. Henrar. M. Biochemistry (Washington) 38 (1999) 4302.W. Pannell. S.W.A. D. Org: Chem. Pomponi.R. O. Kobayashi. K. B: Sci. Ford. Abst. [34] W. Ohyabu. Tackett. Bai. M. Seigler. 111 (1989) 2582. [36] K. N. P. D. Chem.J. S. S. Beart.M. M. Chem.S. Sci. Wright. 51 (1997) 1042. Matsunaga. De-Silva. Tetraedron 54 (1998) 3043. Lavie. Keifer. Huges. L. p. Am. Bowden. Nat. D. Pt. J. Aoki.R. D. B. Watters. Rinehart. [43] H. Vervoort. Marshall. Chemother. Ishibashi. Schmidt. Hamel. Am. J. Sackett. Diss.J. [42] S. A. P. Gäde. From another side. B. Harper. D. J.L. Matsunaga. J. S. Org.W. S. Prod. M. G. Matsumoto. Toxicon 39 (2001) 259. J. Crews. Pomponi. will speed up drastically the discovery of novel active peptides from marine sources. Brune. K. in: D. [32] T. Inc.E. vol. M. McCarthy.R. Gulavita. De Vries. He. Hirota. J. Org. Li. Diss. Crews. Sakai. Clark. [30] K. R.H. The pigment complexes from each major column are pooled and then loaded onto linear sucrose density gradients and centrifuged. H. Sanders.L.J.J. Bewley.. W. J. Horibe. M. Soc. Boyd.A. Lassota.H. 171 (1992) 236. B. as well as in the pharmacological evaluation.K. Int. 118 (1996) 4314. Aneiros. Soc.J. Nat. Diss. Pharmaceutical and Bioactive Natural Products. Longley. Soc. Marine Biotechnology. Nat. Epifanio. 33 (1990) 1634. Nagasawa. McKee.-C.J. Pharmacol. S. 59 (1996) 163. Bewley. Antimicrob. Pt. Chem. [16] C. (September–October) (1997) 6. Faulkner.516. P. 63 (2000) 956. Gustafson. 64 (2001) 117. Lab. Chem. 38 (1995) 338. Lavin. Sakai.H.P. Taylor. Attaway. R.g. P. Shigemori.J. Rinehart. R. these compounds seem to be very useful and promising for biomedical research to clarify many normal and pathological mechanism of action in the human body as well as in the design of very specific and potent new pharmaceuticals for a wide variety of diseases. H. 58 (1997) 3037. J. S. Abst. [35] F. Chem. Rashid. Med. Chem.F. Org.K. Ind. H.J. Mastbergen. L. M. Boswell. [21] R. Oncol. R. Pt. R. J. Nat. [26] J. 1. Rashid. P.W. J. K.A. Faulkner. 44 (1999) 312.J. Sci.).C. 58 (1998) 5403. Prod. Kurosu. Itagaki. Soc. K. Takahashi. G. Another purification procedure in a large preparative level using also an anionic chromatographic column of DEAE cellulose. Huggins. Karai. D. D. Shigematsu. Shield. Weiss. J. Holt.C. [20] J. Fisher. Chem.R.C. L. [38] P. L. Sugawara. McKee. N. Park. K.A. many of these molecules can be found only in a single source. N.A. Pharmacol. Williams. N.G. (July–August) (1987) 136. Mol. in: D. Immunol. Garateix / J.G. Tetrahedron Lett. Schmidt. M.J. Schmidt. Williams. Int. Coleman. Microbiol. Keifer. W. R.H. US Patent 5. Prod. H. Nat.J. 59 (1994) 4849. Gustafson. 56 (1993) 260.F.L.L. S. S.H. N. J. Cichacz. TIPS 16 (1995) 275. S. Cartner.755 (1996). Ciba Found Symp.R. T. Gustafson. Matsunaga. Z.I.K. Fregeau. S. Matsunaga.A. Pannier. M. Zaborsky (Eds. S. A. J. e. J. T. Diss. 16 (1996) 134. Thompson. and sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE) is used to obtain B-phycoerythrin and R-phycocyanin from a microalga [156]. Konosu. [22] P.A. Chem. [33] E. A. O.W. Tartar. M. Matsunaga. Abst. L. Technol.J. Farias. Ogura. 24 (1997) 156. Fusetani. K. J. Drobecq. B 803 (2004) 41–53 51 an appropriate buffer containing mercaptoethanol. Cragg. Lloydia 57 (1994) 79. J. A. B. W. Emrich. F. S. Fusetani. Chem. 5. D. Fusetani. Hawkins. Zaborsky (Eds. K. An alternative procedure to purify R-phycoerythrin from a Mediterranean red algae [157] propose the use of hydroxyapatite produced in the laboratory that can be used batch-wise with large amounts of extracts and therefore can be scaled up. J. [23] C. Schmitz. Attaway.I. exact description of the molecular weight and the sequence. 65 (2000) 782. Hori. Semin. Miljanich.L. M. C. S. New York. Pharmaceutical and Bioactive Natural Products. K. Takebayashi. [31] E. vol.M. P.W. Hashimoto. Concluding remarks Peptidic compounds analyzed here are obtained from very different marine organisms exhibiting different chemical structures and displaying a large variety of pharmacological effects on specific targets. J.L. [13] H. Gerwick. L.A. Eng. Nat. They have been produced along of millions of years of evolution in response of the necessity to evolve in a very competitive environment. [29] J. W. Dorsaz. Pure fractions of phycoerythrin. [28] N. Matsunaga. Bewley. E. Faulkner. Fusetani. D.G. W. M. Maurizi. Boyd. [11] Harbor Branch Oceanographic Institute.E. Barkoczy.J. Fusetani.L.G. [15] E. Schmitz. Wilson.R. 59 (1998) 2200. Prod. D. 60 (1997) 779. [12] S. Toth. [41] C.H. J.A. Martin. A. A multidisciplinary and cooperative effort with the use of more sensitive and fast methods in the analysis of the structure of peptides. Eng. Newman. Toxin 5 (1996) 225.L. R. [25] M.C. Tetrahedron Lett.A.B. Faulkner. I.M. Perun Jr. Chromatogr. Pharm. D. Cancer Chemother.G. J. [37] A. J. Eng. [8] [9] [10] S. Biol.E. S. New York. Béress. Norton. A. [59] S. Pettit.P. R. S. 275 (2000) 35335. G. J. Hasson. Garcia. Gendeh. [106] A. Chávez. D. Béress. Grissmer. Stocklin. S. J. E. J. Perspect. Verdier-Pinard. Fernández-Suárez. Crest. Vita. V. p. E. Patent SNX-111. Hellman. L. Kitahara.I. Biochem. A. [45] G. J. Perspectives in Molecular Toxicology.B. R.R.A. Chavez. S.M.A. [44] G. J.L. Bruhn. Debitus.J. Béress. Jeyaseelan. Engstrom. Aneiros. Biol. Catterall. Cruz. I. R. C. J. Béress. Riguera. Eur. F.52 A.J. Hernández. W. 375.. Fed. McIntosh. Weinstedt. Menez. A.A. Nielsen.R. Pennington. Kelso. L. V. Drug Discov. Heubach.L. [86] J. . U. [100] B. Blumenthal. Martinez. Gomez. Chavez. Harvey. 273 (1998) 32697. Gangwar. Ishida. A. Benoit. Khera. T. 15 (1999) 111.W. L. A. D. Imperial. P. J. [96] W. Acta 71157 (1993) 86.F. J. A. Gurevitz.L. Inc. K. Doubek. J. Seances Soc. Lloydia 59 (1996) 629. Garateix / J. Biochem. Spira. J. Vázquez. Chem. Hessinger. C.R. Biochem. [99] T. Pettit. Des. Rev.M. Miljanich. E. A. Int. Acta 1478 (2000) 9. Qu.I.C. Gulyas. D. B. [64] A. Macek. Justice. A. [104] B. Methods and Tools in Biosciences and Medicine. Shmidt. Pharmacol. Karlsson. Savarin. Rauer. T. [79] A.R. Jones. J. J. 265 (1990) 17141. R. Tuinman. 64 (1995) 493. C. W. Biochem. Antuch. A. A. Beck. 6 (2000) 1293. Marine Biotechnology. Khoo. Gust.A. P. M. A. K.A. J. 118 (1996) 11635. Karlsson. Toxicon 29 (1991) 1051. K. L. McCabe.R. M. D.R. R.J. R. T. I. J. A.A.T. Ramilo. P. Ocampo. Curr. Tytgat. Gilquin. Nadasdi. Jacobsen. F. [50] K. Craig. Science 249 (1990) 257. Castañeda. The Biology of the Nematocysts. Biochemistry (United States) 37 (1998) 5407. Chung. in: D. Bulaj. Blumenthal. C. [84] A. Benoit. V. Qu´ınoa. McKlure. Comp. [78] R. J. Wrischt. [93] C.J. Olivera. Lazdunski. [69] Assignee Neurex Corporation Inventor(s). 277 (2002) 37406. Chem. Padron. Annu.T. Res. J. [97] D. Artiles.J. Pharmaceutical and Bioactive Natural Products. C. T. [71] L. Y. 1. Brain Res.G. E. 244 (1997) 192.S. R. M. Exposito. [87] W.L. J. C. E. Proc.L. R.D. Schmidtmayer. E. [60] L. C. I. M. [55] W. Luchian. D. 44 (1981) 482. L. Newman. Fil. 27 (1971) 117. M. Molgo. Toxicon 37 (1999) 1779. Cruz. D.D. Roberts (Eds. 134 (2001) 1195. J. Tetraedron 54 (1998) 7539. Forest. Adams. Plenum Press. Olivera. [47] R. Pazos. Stessman. S.A. [70] P. Singh. J. Pettit. Kizu. Biol. Kamano.P. Alewood.). Aneiros. [67] R. E.J. Byrnes. Thomas. Y. Cell Biol. R. Adams. De Medeiros. R. Hillyard. Abogadie. Toxicon 33 (1995) 605. G.C. [85] W. R. M. Kem. Sampieri. M. D. Lanigan. Chua. Nicholson. Grissmer. Fernández. Chem. C.M. J. J. Harvey. Chem. A. Alvarez. Zaborsky (Eds. Lamthanh. Biochemistry 36 (1997) 11461.J. M. Quiñoa. H. A. 271 (1996) 9422. [76] L. Tejuca.G. Salceda. Mol. Huerta.W. Gray. M. E. Schmidt. Brown. Jones. R. Chem. C.A. Chem. Zaydenberg. Visan. Sotolongo. Marshall. G. Clark. Gray. Paschetto. R. [49] R. Des. Inoue. Chem. C. Chromatogr. E. M. J. [48] T. Gordon. Biol. J. Schmidt. J. R. R. C. M. R. Akhtar. Guenneugues. Norton. Tomer. 36 (1995) 8853. Grotendorst. Basada. J. G. Menez (Ed. C. Nguyen. Biochem. [51] F. 303 (2002) 1067. Rochat.R. W. Hessinger. Mènez. Gasparini. Principles of Toxicology: Environmental and industrial Applications. Am. [62] J.P. K. Adams. J. Y. Riguera. C. J. Tarczy-Hornoch. J. Cancer Res. Chem. J. Animal Toxins. Rogers. Christina. J. P. 32 (2000) 1017. M.C. E. Dutton.B. C. W. Gilles. Cebada. Garateix. J.J. O. L. S. Pennington. L.P. Schulze. Petitt. J. [91] E. V. Chung. Biol. Biochim. Olivera. M. [75] G. E. C. Debitus. Ménez. New York. K. FEBS Lett. B. Y. Prod. Comp. D. K. [82] G. D.L.T. Toxicon 5 (1968) 253. Tanada. Delfin. B 803 (2004) 41–53 [73] M. Huie. 427 (1998) 149. Anderson. J. Kamano. M.R. M. Khaytin. Tetraedron Lett. Blanton. C. Caterall. Schweitz. Kem.M. J. 270 (1995) 16796. [61] G. Med. Schaller.R. Olivera. B. B. M. Engstrom. G. DDT 5 (2000) 98. [101] P. second ed. 287. Herald. England. Petitt. Ther. Am. D. Gohil.M. Aneiros. Li. Lotan.L. M..E. 192 (1998) 409. A.C. Neurophysiol. Abbruzzese. Michel. R. M. Corpuz.V. 409. Bontems. Castellanos. D. Herald.S. G. Romero. S. R. Lanio.A.M. D.C. Pharmacol. R. Gola. 6 (2000) 1249. Craik.J. Iglesias. H. Guillemare.M. Yamashiro. Kizu. Mart´ın-Eauclare.A. Biochem. Madden. Ramachandran. J.L. Shon. 2000. E. Possani. [83] S.L.L. Hamel. Biol. I.T. K. Pharmacol. Bond. A. Am. Harvey. L. [66] B. Gonzalez. H. M. Debitus. Mena. S.). Prod. Bertrand. Riguera. Ramachandran. P. A.M. C. Exp. p. H. K. 1993. K. J. Soto. A. Martinez. 2000.C. [88] C. Moinier. E. T. López. I.J.).L.E. K. V. [52] R. Vega. B. Zinn-Justin. Andrews. 15 (1993) 675. Mansuelle. Biochim. Gutman. Ferrer. [92] G. Vázquez. Wuthrich.J. Julius. Aneiros.M. N. Annu. M. [94] E. in: D. [56] R. Toxicon 36 (1998) 41. J. C. Physiol. Proc.P. Number 5364842 Issue Date 1994 11 15 Appl. [63] H.-F. D. Aneiros. 63 (1994) 823. Racapé. Biol. [72] Y.A. 74. J. Galán. J. Shichor. Norton. Hamel.D. F. Cell. McIntosh. Shen. Gasparini. Drum.R.L. M. Olivera. Castañeda. T. Y.L. Toth. Garateix. Imperial. E.L. D. L. Salceda. B. Physiol.E. K. E. [46] G. Baczynskyj. Iglesias. Bai. [95] E.D. K. P. E. Mahnir. [102] J. K. M.E. Cotton.A. Dannmeier.M. Soc. Woodward. Lenho (Eds. Dufresne. Minagawa. Lecoq. J. Fuji. K. P. Drinkwater. Eckhardt.L. Wright. Hillyard. M. Y. Craig. M. Goudet.R. E.R. Biol. Batista. Menéndez-Soto Del Valle. [65] J. Shiomi. Boettner. G. Shapiro. H. A. 25 (1994) 199. Marsh. in: H. Br. Bowden. 59 (2001) 462. Muñoz. [105] M. G.M. Sausville. A. O. D.M. J.S. K. Kem. [80] O. 269 (1994) 16785.L. J.M. [103] K. Jiménez. Chem. C. Tran.N. M. [81] H. Sharpe. Attaway. Alessandri. [54] M. U. Nat.J. C. Sánchez Rodriguez. 109 (1987) 6883. T. Amor.R.M.R. C. D. Spira. 2002.H.M. Herald. G. [89] B. in: A. K. Fed. Layer. Anderson. 103 (1992) 403. A. Arias.J.M. Bai. Delfin. Harvey. 40 (1) (1981) 21. John Wiley and Sons.M. Blumenthal. [57] B. Fernández. V. A.A. K. P. [53] M. Norton. Rivier.L. Soc. [58] B. A.F. Pharm. L. A 185 (1999) 353. Toxicon 39 (2001) 693. Pennington. Hart.R. p.A. H.W. Bouet. T. I. Anderluh. W. Biochem. p. Más. T. Young. Rivier. E. R. Hanck. Data 081863 1993 06 23. [90] R. Ravens. [74] G. Morera. Lancelin.J. R. R. 215. Lewis. Sauviat. 32 (2001) 1017. Biochemistry 34 (1995) 8076. S. Wang. J.L. James. Gilquin. Chavez. USA. Arch. M Schmidt. L. p.M. Heinemamm. Bruhn. Qu´ınoa. Nat. 864 (2000) 312. Dias-Kadambi. D. J. A.L. 111 (1989) 5015. BioScience 47 (1997) 131. Wesson. Valentino.A. Science 281 (1998) 575. Chem. Biophys. Eur. [77] W. Chem.J. Kamano.J. S. Loughnan.H. [98] J. Kallen. Mahnir. R. Bates. J. Schmitz. Biol. M. C. Soc. H. Biol. Clin.S. Toxicon 40 (2002) 111. Rochat.M. Chem. Kem.L. Wachter. J. J. H.J. Garateix. T. 1988. Toxicon 39 (2001) 187.F.K. Hasson. M.S. M. M. Cerny. Nagashima.F. Academic Press. Soto. 73 (1995) 1295. M.R. Cahalan. Kem. Olivera. Alvarez.). Berndt. N. V. D. D. in: P. Biochemistry 35 (1996) 16407. H. Loret. [68] L. vol. Kalman.R. Rodr´ıguez. Herald. Kiel. C. J.L. Chandy. F.E. J. Matheson. W. Biol. Diaz.M. Int. A. 271 (1996) 15959. Hamann. 42 (1983) 101. M. Biophys. 270 (1995) 25121. Scheuer. A. Williams. Pusztai. Rev. Alsen. Biochemistry 35 (1996) 3503.R.E. A. Meeresforsch.S. K. Y. Karlsson. Adriaenssens. Chem. Morera. Biotech.W. G. Barlic. L. B. Alvarez. [118] P. Betancourt. Lazdunski. W.S. [116] M. Appl. Vyazovkina. Gómez. Biophys. Cochin India Society of Fisheries Technologists.N. Adv.V. Young.-N. Béress.P. Calvette.H. R. K. M. B. Menestrina.B.C.). M. Biophys. Ch. Padrón. G. P. G.N. Jacobs. Khalilov.A. P. 264 (1989) 1. T. Mekideche. Macek. M.J. Glazer. 3 (1996) 317.V. Cline. F. Freitas. Artiles. G. [108] M. McGough. Strichartz (Eds. E. M. Possani. Chavez.M.A. Sampaio. R. Exp. B. 17 (1998) 18.W. M. Tytgat. Zaydenberg. F. A. Hall. [115] P. Org. E.P.N. Bruhn. T. J. Biol. Molnar. Toxin Rev. E.M. Klottz. 304.P. Biochem. Arch. Macek.M. Hill. J. Malovrh. Wunderer. Res. Alvarez. Chem. K. Lanio. V. McIntosh. Burnet. M. A. 2000. Pennington. in: M. Trends Immuno-Haematol. Animal Toxins. G. F. Tejuca. Sánchez-Rodr´ıguez. J. K. L. Pauron. 93 (2002) 73. Dalla Serra.E. Creppy. Nagabhushanam (Eds. Antony. [129] J. 234 (1995) 329. in: K. D. M.W. [152] J. Biochim. [157] R. Phytochemistry 27 (1988) 2063. [123] E. Cruz. Diochot. P. Membr. S. Badakere. [150] G. Adv. Chromatogr. 11 (1997) 348. Kumpf. Calton. S. Gray. Mod. 2 (1994) 92. Chem. Bernheimer. Murciano. B 803 (2004) 41–53 [107] H.A.S. [137] M. Apt. Goudet. Chem. Bidarrd.M. Menestrina. Gray. 5 (1988) 830 (in Russian). Bernheimer. Layer. Sarajoni. Bulaj. Mol. Glazer. Photosynth. J.M. Tan. J.H.I. Zecchini.F. [110] S.E. J.) Nutrients and Bioactive Substances in Aquatic Organisms. Pennington.).M. Biochim. Biochem.). Williamson. H. 6 (1994) 105. Aneiros. J. R. J. Biochemistry 35 (1996) 14947. 273 (1998) 6744. Vilbois. Biotechnol. J. Morse. Res. Frelin. Hunkapiller. G. Silveira. J. 346 (2000) 223. I. [117] T. Acta 1159 (1992) 1. D. A. Methods and Tools in Biosciences and Medicine. Monastyrnaya.F. Elyakov.M. N. C. 1990. DLI-Press.E. Phycol. Bhatia. Pederzolli. M.S.E.R. structure and molecular pharmacology.-C. B.. Dalla-Serra. Norton. G. Klotz. Johnson.T. Cruz. Tejuca. De Chastonay. 220 (1996) 4372. Biophys. Pallaghy. A. M.T. D. C. Biol. Nair. [139] F. M. M. 57 (2000) 343.A. D’Ambrosio. Eur. H. [112] T. Wagstaff. Biophys. R. Ungaro. Chem. Hori. Ramos. [127] J.O. Birkhauser Verlag.P. Martin-Euclarie (Eds. Samuel.R. Béress. Joseph (Eds. Acta 667 (1981) 87. Smith.D. R. Konya. Devadasan. A. Biol. Lagomasino. Gorczyca. Toxicology 87 (1994) 205. Cell. Toxicon 26 (1988) 997.V. O. G. Perigreen. Molina.J. [142] R. P. A. p. Washington. Kozlovskaya.M. Chuy.M. Macek.J. A. Ivanov. Krafte. J.J. [154] J. 101 (2003) 289. p. Pharmacol. T. Schweitz. M. Grossman. 273 (1995) 354.T. Passaquet. Leyva. Biophys. [109] J. Wachter. 218 (1982) 329.S.K. Biol. C. 1997. Menestrina.L. Olivera. Menestrina. K. H. Corpuz. C. S. D. J. [141] K. G.M. W. [148] A.A. 1991.M. J. R. McIntosh. 275 (2000) 32391. Res. A. in: An Indo-United States Symposium. Zykova. W. 10 (1991) 223. L. M. Hoppe-Seyler 370 (1994) 115. B. Br. p. Podlesek.D. G. Béress. India. A. D. Sidler. Khaytin.K.N. R. W. A. [132] L. B. [140] J. Toxicol. Pazos. Res. Biochem. W. 253 (1978) 8303. Monastyrnaya. M. Chem 274 (1999) 2472. J. Toxicon 15 (1977) 177. L. Belmonte. G. G. [114] P. Chablani. An Approach to Protein Chemistry. Miyazawa. Mendoza. Olivera. Menestrina.A. M. I. Biochim. Batista. Zuber.E. Thompson. I. [138] H. D. Nagano. Garret. p.S. Switzerland. Mahasneh. A.V. C.B. Thomas.M. Rossano.M. A. Betancourt. Chem. 1994. Luth. [125] A. Turk. [136] W.F. 28 (2000) 419. [134] M. Ikegami. Lagomasino. 16. Saboureau. [155] G. L. M. Williams. [135] C. J. R. Lanio. J. Tsugita. Ennamany. Glazer. M. Pennington. Vickneshwara. A.E. Biotech. [131] H. L. Pungerèar. Biochem. L. Facts and Protocols. Gerwick. Schweitz. Acta 987 (1989) 181. Dalla-Serra. Khim. Chem. [145] W. Garg. Ito. in: S. I. T. Béress. 3 (1995) 78.R. Zecchini. P. 32 (Chapter 3).A. Riccio.R.R. Olivera.J. L. W.E. Aneiros. J. Cruz. [146] K. Handbook of Experimental Immunology. Pharmacol. A. E.D. F. R. G. Alvarez-Pez. Biol. K. Ch. Schweitz. [113] J. Acta 541 (1978) 96. Areces. Mod. Hessinger.H.M.R. Biol. Byrnes. Oxford. Life Sci.B. [120] P. Klotz. Doyle. Biol. Anderluh. Biotechnol. Phytother. [121] G. Volberg. Vijverberg. Strukelj. Aneiros. F. J. Alvarez. J. J. 274 (1999) 21885. Biochi. Chem. [124] A. B. Strupat.I.D. [122] E. Toxicol. 53 . V.W. Bioorg. McIntosh. Biol. Elliot. J. J. 12 (1982) 101. Cline. Biol. Mukundan. D. Mendoza. A. 134 (2001) 1195. L.T. A. Garateix.P. in: M. J. DC. Kem. T. C.). Biol. Mart´ınez. K. J. Bermejo. M. [151] M. J. in: H. Holborow (1986). 1986.G.C. H. M. Kem. Comments Toxicology 9 (2003) 1 (in press). [156] R. Smith (Eds.J. F. Gubensek. Toxicon 40 (1995) 234.M. Biophys. Acien. B. Ferrer. L.V. Areces. Besada. Lagomasino. Glazer. Tamkun.J. [153] J. Apalikova. American Chemical Society. E. J. Commun. Tudor. Wiebe. Biol. Bloch Jr.E. Nat. Suter. [126] M. P.W. Liuzzi. V. J. Costa. R. M. Blackwell Scientific Publications. Perez. J.M. J. H. Cavada. [128] J. Watts. J. E. [147] A. Ferreras.W. Tejuca. California. J.J. Biochem 24 (1985) 3554. Kozlovskaya. J. Avigad. Mahnir. Toxicon 13 (1975) 359. Rochat. [133] J. Z. Toxicon 24 (1986) 117. 32 (1995a) 309. Lanio. Pederzolli. V. A. Lagomasino. C. Huerta. 135 (1995) 235. Klottz. M. [130] L. Saker-Sampaio.D. Hum. M. Galán. G. Rauer. Alvarez.F. [111] R. A. L. N. A.S. Garateix / J.V. G. E. Lagomasino. [149] S. Toxicon 39 (2001) 187. Bretting. Macek. Macek. [143] A.S. [144] L. Pazos. Lazdunski. 24 (1998) 509. V. A.M. Yamanaka. Bioactive compounds from marine organisms with emphasis on the Indian Ocean. 167. Basel. Chem. [119] C. Cahalan. Alvarez. Yoshikami. Anderluh.M.I. Grangeiro.W. Dunn. H.M.G.C. Marine toxins: origin. Struct. Buck. M. J. R. L.M.A. G. 259 (1984) 14343. A.A. Shvets. V.S.